초록

○ 연구결과
1) 단백질 분해효소의 작용 조건에 따른 우유단백질의 분해 특성 분석 및 분리 정제
2) 활성 유청단백질 분해물 제조공정 최적화 및 대량생산공정 정립
3) 활성 유청단백질 분해물의 물리화학적 특성 구명 및 관능적 특성 평가
4) 단백질 분해효소 생산 미생물 선발 및 분해물의 분별
5) 조골세포 배양 실험을 이용한 유단백질 분해물의 생체 외 성장 촉진 효과 분석
6) 동물실험을 이용한 유단백질 분해물의 생체 내 골밀도 증진 효과 분석

Abstract ▼

Milk is a rich source of nutritional components and contains biologically active substances. In addition, various latent bioactivities of peptides are generated by digestion, enzymatic hydrolysis or fermentation. Physiological peptide derived from milk with scientific evidence have been described in

Milk is a rich source of nutritional components and contains biologically active substances. In addition, various latent bioactivities of peptides are generated by digestion, enzymatic hydrolysis or fermentation. Physiological peptide derived from milk with scientific evidence have been described in the literature and some of them are available in the markets.
we have focused on the bioactivity related to bone health since osteoporosis is a major health problem in many countries including Korea.
Therefore, this study has been conducted to develop processing technology for the production of bone density enhancing milk-derived peptide.
Among various enzymatic hydrolyates, basic fractions of whey protein hydrolysate produced by Flavourzyme (5% based on protein content) showed the highest growth promoting activity for osteoblastic MC3T3-E1 cells. The optimum hydrolysis temperature and time predicted by response surface methodology was 54℃ and 140 min, respectively. At this hydrolysis condition, 39% of whey protein was hydrolyzed and 79 peptides were generated. The estimated average peptide chain length and 2.8.
Depending on the conditions for enzyme inactivation, the growth promoting activity for osteoblastic cells in whey protein hydrolysate was changed. The growth promoting activity in whey protein hydrolysate was heat resistant (85℃, 5 min) and remained in the soluble fraction at pH 4. The active peptide fraction was effectively recovered in the soluble fraction by ethanol precipiatation (30%, w/w) and the recovered activity was close to that of basic fraction obtained by cation exchange column chromatography. Based on the result of reverse phase HPLC, ethanol treatment removed hydrophobic peptides from the hydrolyates. This results suggest that the active component can be concentrated by the treatment of heat and ethanol and can be used for rapid large scale concentration process.
The whey protein hydrolyate showed excellent solubility and turbidity in wide ranges of pHs whereas it had reduced emulsifying activity. The sensory quality of whey protein hydrolysate (0.5 and 1%) was examined using commercial amino acid drink as a carrier. There were no significant differences in the intensities of sensory attributes including sweetness, bitterness, saltiness and umami. However, significantly reduced sourness was observed in the samples containing whey protein hydrolysate. This result was due to the basic amino acids contained in the samples and the samples containing whey protein hydrolysate showed significantly higher pH than that of control.
The growth promoting activity for osteoblastic MC3T3-E1 cells in whey protein hydrolysate were not significantly changed up to 1 month when the hydrolysate was stored at 4℃.
The proteolytic activity of various probiotic bacteria were examined and three probiotic strains (Lactobacillus casei ATCC 393, Lactobacillus acidophilus ATCC 4356 and Lactococcus 449) were selected based on production of non-protein nitrogen (NPN) and free amino nitrogen (FAN). They showed high growth and proteolytic activity when they were incubated at 37℃ for 15 hr in the medium containing 12% of SNF. At this condition, generation of small peptide about 1 Kda were observed.
The growth promoting activity of milk protein fermentation products for osteroblastic MC3T3-E1 cells was tested. Among the fermentation products, the products by Lactobacillus casei ATCC 393 (LC-393) showed the highest activity. The significantly higher levels of biochemical markers (alkaline phosphatase activity and osteocalcin mRNA expression) indicating enhanced osteoblastic cell proliferation and differentiation was found when the whey protein hydrolysate (WPC-H) or fermentation products were added. In addition, apoptosis of MC3T3-E1 cell were significantly inhibited in the presence of WPC-H or LC-393.
The changes in protein profiles of MC3T3-E1 by the exposure of LC-393 were examined by two dimensional gel electrophoresis. Total ten protein spots with three-fold increase or decrease were observed. Among them, four proteins were identified by MALDI-TOF. Especially, Eva6 protein exhibited high homology with fibroblast growth factor (FGF) receptor 3. Recent reports suggest that FGF was one of growth factors for inducing the proliferation of osteoblastic cells.
The effects of WPC-H or LC-393 on bone metabolism were investigated using the ovariectomized (OVX) rats. The final body weight, food intake and food efficiency did not show significant differences among treatment groups except sham group. The BMD (bone mineral density) and bone strength of ovariectomized rats were significantly improved (p<0.05) by the supplementation of WPC-H (2%) or LC-393 (2%). The activity of bone biochemical markers also increased in the group containing either WPC-H or LC-393. The concentration of C-telopeptide collagen type I, the bone resorption marker was significantly decreased (p<0.05) in the group containing either WPC-H or LC-393. Based on the result of dosage trial, less than 1% of WPC-H or LC-393 supplementation was enough to enhance bone density.

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 요약문 ... 3
• SUMMARY ... 10
• CONTENTS ... 13
• 목차 ... 14
• 제1장 연구개발과제의 개요 ... 15
• 제1절 연구개발의 필요성 ... 16
• 제2절 연구개발의 목적 및 범위 ... 17
• 제2장 국내외 기술개발 현황 ... 18
• 제3장 연구개발수행 내용 및 결과 ... 20
• 제 1 절 주관과제 : 우유단백질의 효소 분해에 의한 가수 분해물의 제조, 물리화학적 특성 구명 및 활성 펩타이드 제조를 위한 대량생산공정의 개발 ... 20
• 제 2 절 협동과제: 미생물 발효에 의한 우유단백질 분해물의 분별 및 생체 내.외에서의 골밀도 증진효과 검증 ... 102
• 제4장 목표달성도 및 관련분야의 기여도 ... 152
• 제 1 절 주관과제 우유단백질의 효소 분해에 의한 가수 분해물의 제조, 물리화학적 특성 구명 및 활성 펩타이드 제조를 위한 대량생산공정의 개발 ... 152
• 제 2 절 협동과제 미생물 발효에 의한 우유단백질 분해물의 분별 및 생체 내.외에서의 골밀도 증진효과 검증 ... 153
• 제5장 연구개발결과의 활용 계획 ... 155
• 제6장 연구개발과정에서 수집한 해외과학기술정보 ... 156
• 제7장 참고문헌 ... 157
• 끝페이지 ... 164